Bacterial Cell Surface Display of a Multifunctional Cellulolytic Enzyme Screened from a Bovine Rumen Metagenomic Resource
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초록
A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.
Abstract
A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.
참고문헌 (26)
- Beguin P, "The biological degradation of cellulose", FEMS Microbiol. Rev, 13, pp.25~58 (1994)
- Clarke AJ, "Biodegradation of Cellulose: Enzymology and Biotechnology", pp.23~68 (1997), Technomic Publishing
- Cornelis P., "Expressing genes in different Escherichia coli compartments", Curr. Opin. Biotechnol, 11, pp.450~454 (2000)
- CHOI, JONG HYUN, "Display of Proteins on the Surface of Escherichia coli by C-Terminal Deletion Fusion to the Salmonella typhimurium OmpC", Journal of Microbiology and Biotechnology, 15(1), pp.141~146 (2005), 한국미생물·생명공학회
- He nrissat B, "Synergism of cellulases from Trichoderma reesei in the degradation of cellulose", Biotechnology, 3, pp.722~726 (1985)
- Ito J, "Regulation of the display ratio of enzymes on the Saccharomyces cerevisiae cell surface by the immunoglobulin G and cellulosomal enzyme binding domains", Appl. Environ. Microbiol, 75, pp.4149~4154 (2009)
- Kim JY, "Mining and identification of a glucosidase family enzyme with high activity toward the plant extract indicant", J. Mol. Catal. B Enzym, 57, pp.284~291 (2009)
- KIM,KYU-YONG, "Display of Bacillus macerans Cyclodextrin Glucanotransferase on Cell Surfaceof Saccharomyces cerevisiae", Journal of Microbiology and Biotechnology, 12(3), pp.411~416 (2002), 한국미생물·생명공학회
- Ko KC, "Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system", J. Microbiol. Methods, 94, pp.311~316 (2013)
- Ko KC, "A novel bifunctional endo-", exo-type cellulose from an anaerobic ruminal bacterium, 2011, pp.89, 1453~1462
- Ko KC, "A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria", Biochem. Biophys. Res. Commun, 441, pp.567~572 (2013)
- Lee JS, "Surface-displayed viral antigens on Salmonella carrier vaccine", Nat. Biotechnol, 18, pp.645~648 (2000)
- Lee SY, "Microbial cell-surface display", Trends Biotechnol, 21, pp.45~52 (2003)
- L iljeqvist S, "Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus", Appl. Environ. Microbiol, 63, pp.2481~2488 (1997)
- Lin L, "Characterization of extracellular cellulose-degrading enzymes from Bacillus thuringiensis strains", Electron. J. Biotechnol, 15, pp.1~7 (2012)
- L ynd LR, "Microbial cellulose utilization: fundamentals and biotechnology", Microbiol. Mol. Biol. Rev, 66, pp.506~577 (2002)
- Park SJ, "Secretory production of recombinant protein by a high cell density culture of a protease negative mutant Escherichia coli strain", Biotechnol. Prog, 15, pp.164~167 (1999)
- P uente JL, "The Salmonella ompC gene: structure and use as a carrier for heterologous sequences", Gene, 156, pp.1~9 (1995)
- Stahl S, "Bacterial surface display: trends and progress", Trends Biotechnol, 15, pp.185~192 (1997)
- Tanaka T, "Recent developments in yeast cell surface display toward extended applications in biotechnology", Appl. Microbiol. Biotechnol, 95, pp.577~591 (2012)
- Tsai SL, "Functional display of complex cellulosomes on the yeast surface via adaptive assembly", ACS Synth. Biol, 2, pp.14~21 (2013)
- Tsai SL, "Functional assembly of minicellulosomes on the Saccharomyces cerevisiae cell surface for cellulose hydrolysis and ethanol production", Appl. Environ. Microbiol, 75, pp.6087~6093 (2009)
- van Bloois E, "Decorating microbes: surface display of proteins on Escherichia coli", Trends Biotechnol, 29, pp.79~86 (2011)
- Wieczorek AS, "Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis", Microb. Cell Fact, 9, pp.69~ (2010)
- Wu CH, "Versatile microbial surface-display for environmental remediation and biofuels production", Trends Microbiol, 16, pp.181~188 (2008)
- Xu Z, "Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif", Appl. Environ. Microbiol, 65, pp.5142~5147 (1999)